Journal: bioRxiv

Article Title: Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems

doi: 10.1101/2025.11.16.688697

Figure Lengend Snippet: (A) Left: Phosphorylation of eIF2α on Ser52 converts eIF2 into an inhibitor of its guanine nucleotide exchange factor (eIF2B), blocking ternary complex regeneration and suppressing translation initiation. Right: Phosphorylation of eEF2 on Thr57 by eEF2 kinase (eEF2K) prevents ribosomal translocation, thereby slowing peptide elongation. (B) Phosphorylation of eIF2α (Ser52) by stress-activated kinases (PKR, GCN2, PERK, HRI) converts active eIF2 into an inhibitor of its guanine nucleotide exchange factor eIF2B, thereby preventing ternary complex formation and suppressing global initiation. (C) The eEF2 cycle: eEF2K phosphorylates eEF2 at Thr57, which prevents ribosomal translocation; dephosphorylation by PP2A restores elongation. (D) Illustration of the experimental workflow for lysate generation and in vitro translation (IVT). Suspension Expi293F cells were harvested, lysed under native conditions, and the resulting extracts programmed with a Nanoluciferase (NanoLuc) reporter mRNA to quantify translational efficiency. (E) Schematic representation of human Expi293F cells engineered by prime editing (PE) to introduce phospho-null substitutions in EIF2S1 (eIF2α S52A) and EEF2 (eEF2 T57A). All amino acid residue numbers correspond to the human reference sequences according to the UniProt database, entries P05198 and P13639 for EIF2S1 and EEF2, respectively. (F) Comparison of translational output in IVT reactions programmed with NanoLuc mRNA using extracts prepared from wild-type Expi293F cells or genome-edited Expi293F eIF2α-S52A and eEF2-T57A lines. All experiments were performed in biological triplicates.

Article Snippet: Antibodies for RPS19 (A304-002A) and eEF2K (A301-686A-T) were purchased from Bethyl Laboratories Inc. Anti-mouse IgG-HRP (sc-525409) and GADD34 (sc-373815) were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Blocking Assay, Translocation Assay, De-Phosphorylation Assay, In Vitro, Suspension, Introduce, Residue, Comparison

Journal: bioRxiv

Article Title: Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems

doi: 10.1101/2025.11.16.688697

Figure Lengend Snippet: (A) Schematic of the EIF2K (encoding eEF2 kinase) genomic locus and CRISPR–Cas9 targeting strategy. Exons and intron structure are shown with the chromosomal position (16p12.2). A single guide RNA (sgRNA) was designed to target exon 3, introducing a frameshift mutation predicted to disrupt kinase catalytic function. Sequence is according to Homo sapience reference genome assembly GRCh38.p14 (GenBank assembly accession: GCA_000001405.29). (B) Validation of eEF2K knockout clones by immunoblotting. Whole-cell lysates from three independent eEF2K-KO clones and wild-type (WT) Expi293F cells were analyzed by Western blot using an anti-eEF2K antibody. All KO clones showed complete loss of eEF2K protein, while eS19 served as a loading control. Gels are representative of two independent experiments. (C) Growth characteristics of eEF2K-KO clones. All three knockout clones proliferated at rates comparable to WT Expi293F cells, indicating that eEF2K loss does not affect suspension-culture viability or growth kinetics.

Article Snippet: Antibodies for RPS19 (A304-002A) and eEF2K (A301-686A-T) were purchased from Bethyl Laboratories Inc. Anti-mouse IgG-HRP (sc-525409) and GADD34 (sc-373815) were from Santa Cruz Biotechnology.

Techniques: CRISPR, Mutagenesis, Sequencing, Biomarker Discovery, Knock-Out, Clone Assay, Western Blot, Control, Suspension

Journal: bioRxiv

Article Title: Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems

doi: 10.1101/2025.11.16.688697

Figure Lengend Snippet: Western blot analysis of extracts prepared from WT, eEF2 T57A mutant, and eEF2K knockout Expi293F cells. Immunoblotting with phospho-specific antibodies shows that eEF2 phosphorylation is completely abolished in both mutant and knockout strains, confirming loss of eEF2K-dependent modification at Thr57. Total eEF2 levels remain unchanged across all samples, as detected by pan-eEF2 antibody. Ribosomal protein eS19 serves as a loading control. Gels are representative of two independent experiments.

Article Snippet: Antibodies for RPS19 (A304-002A) and eEF2K (A301-686A-T) were purchased from Bethyl Laboratories Inc. Anti-mouse IgG-HRP (sc-525409) and GADD34 (sc-373815) were from Santa Cruz Biotechnology.

Techniques: Western Blot, Mutagenesis, Knock-Out, Phospho-proteomics, Modification, Control